The pathology of breast cancer is frequently complicated by severe and lethal hypercalcemia. A humoral mechanism for hypercalcemia is operative in more than half of breast cancer patients. The long-term objective of our research is the control of breast cancer-associated humoral hypercalcemia of malignancy (HHM) mediated by the parathyroid hormone-related protein (PTHrP). Vitamin D3 downregulates the PTHrP gene and also inhibits cell proliferation, properties that qualify vitamin D3 as a potentially useful agent in the treatment of breast cancer, including the control of PTHrP- mediated HHM. In this project, the mechanism by which vitamin D3 represses PTHrP will be defined. The human PTHrP gene is a complex transcriptional unit, utilizing at least three different promoters. Different cell lines and tissues exhibit different promoter utilization patterns. Using transient transfection assays, the responsiveness of each promoter to vitamin D3 will be assessed in cell lines derived from normal tissue and breast tumors. The precise sequence elements conferring responsiveness to vitamin D3 within a particular promoter will be located by deletion mapping. Trans-acting nuclear proteins interacting with these promoter elements will be characterized by gel retardation and nuclease protection assays. The hypercalcemic effects of vitamin D3 itself have thus far prevented its clinical application. Recently, various non-hypercalcemic vitamin D3 analogues have been synthesized. Nonhypercalcemic vitamin D3 analogues will be tested for their ability to (i) down-regulate PTHrP gene expression and decrease PTHrP secretion, and (ii) inhibit cell proliferation, thereby addressing whether these derivatives exert the same net desirable effects as the parent compound and therefore are of potential value therapeutically. PTHrP has been classified as a member of the immediate early gene family, and has growth factor-like properties. Therefore, the effect of PTHrP on breast cell proliferation will be examined, using antisense oligonucleotide technology or, alternatively, antisense RNA technology. These studies will form a molecular basis towards the clinical application of non-hypercalcemic vitamin D3 analogues as therapeutic agents targeted at both PTHrP-mediated HHM and at cell proliferation.